Despite the implementation of childhood vaccination schemes and the availability of effective antibacterial agents, bacterial meningitis remains to affect individuals in developing and developed countries. Meningitis is a central nervous system (CNS) infection often associated with severe outcomes and significant long-term effects in a substantial number of survivors. This disease thus requires prompt diagnosis and treatment (1). Complications may be sudden or gradual in onset and may arise anytime after the appearance of initial symptoms, including the time after completion of therapy. Symptoms of meningitis include fever, headache, confusion and vomiting, often preceded by symptoms of an upper respiratory tract infection. Clinical features used for diagnosis include cerebrospinal fluid (CSF) pleocytosis with predominance of neutrophils, elevated CSF protein, decreased CSF glucose and isolation of the bacteria from the CSF by culture. S. pneumoniae, Neisseria meningitidis and Haemophilus influenzae are the major bacterial pathogens causing meningitis with S. pneumoniae being responsible for two thirds of meningitis cases in the developed world (1). S. pneumoniae is a Gram-positive pathogen, which colonizes the nasopharyngeal mucosa of children as well as adults and is transferred among individuals by coughing and sneezing (for review, see 2). In addition to meningitis, S. pneumoniae causes a broad spectrum of diseases, including otitis media, pneumonia and bacteremia (2), leading to substantial morbidity and mortality worldwide. Mortality due to pneumococcal meningitis ranges between 15 and 40%, while ~50% of survivors experience long-term health effects. Of note, even after a good recovery, neuropsychological testing demonstrates cognitive impairment in approximately one third of survivors (3,4). Vaccination with the pneumococcal conjugate vaccine has reduced pneumococcal invasive diseases caused by the serotypes included in the vaccine, but this reduction has been offset by increases of carriage and disease caused by strains not included in the vaccine formulation (2,5,6). The following review highlights the underlying mechanisms of neural complications following CNS invasion of S. pneumoniae.

The pneumococcus habitat is the human nasopharynx mucosa with a prevalence of ~40% in infants and 15% in adults (7). The bacterium is transferred between individuals mainly by coughing and sneezing. Once in the niche, bacterial survival depends on adherence, nutrition and replication; the pneumococcus has to overcome the host's immune system as well as other microbial species that can colonize the same niche (8). Two types of pili identified in S. pneumoniae are responsible for initial attachment of the bacterium to the host. The first pilus is an oligomeric appendage encoded by the rlrA operon (9) and the second pilus is encoded by pilus islet 2 (10). To reach the epithelial cell layer and colonize the nasopharynx, S. pneumoniae degrades the mucus by exoglycosidases such as neuraminidase A, β-galactosidase, β-N-acetylglucosaminidase and neuraminidase B, decreasing mucus viscosity and preventing mucus entrapment (11). In addition, it produces pneumolysin, a toxin that decreases epithelial cell cilia beating and enhances bacterial adherence (12). Pneumococcus also expresses the enzymes N-acetylglucosamine-deacetylase A and O-acetyltransferase that provide resistance to lysozyme that cleaves the peptidoglycan in the cell wall of the pathogen (13). To compete with the host innate immune system at the nasopharynx site, the pneumococcus has developed several strategies: i) Encapsulation and the use of proteases which prevent binding of secretory immunoglobulin (Ig) A and cleave IgA antibodies, immune system components designed to facilitate phagocytosis of the bacteria (14). ii) Inhibition of the activity of Lactoferrin, an iron-binding glycoprotein, thus enabling the utilization of iron, which is necessary for the bacteria's metabolism (15). iii) Limitation of the activation of the complement cascade which normally promotes the cleavage of several complement factors, leading to bacterial opsonization and phagocytosis, leukocyte recruitment and creation of pores in the pathogen's membrane, thus inducing cell lysis (6,16).

Following the initial attachment, adhesive molecules embedded in the bacterial cell wall or the cytoplasmic membrane are exposed as a result of shedding of the capsule (17). Among these adhesins are: The lipoprotein pneumococcal surface adhesin A (18) which binds to the E-cadherin receptor (19); the Pneumococcal adherence and virulence factor (Pav)-A protein, which binds to the extracellular matrix protein fibronectin to produce a Pav-A-finbronectin complex that binds an integrin receptor (20,21); fructose bisphosphate aldolase (22,23), which was found to bind the flamingo cadherin receptor and NADH oxidase, which was found to bind, among others, laminin α5 and contactin 4 (24). Recently, Pav-B and CbpA (CbpA, also known as PspC) were found to have an important role in the interaction of S. pneumoniae with matricellular glycoprotein thrombosphondin-1, a mediator of bacterial adhesion to host cells (25).

After binding, S. pneumoniae interacts with additional adhesins. These include phosphorylcholine, which binds to the platelet activating factor receptor (PAF-R), CbpA (26) which binds to either the polymeric Ig receptor (pIgR) or to secretory IgA (21). The latter two adhesins are considered invasins, since they facilitate transcytosis through the mucosal epithelial cell layer from the apical membrane to the basal membrane using the PAF-R and pIgR recycling pathways (27). Binding and crossing the epithelial cell layers allows access to the submucosal layer, leading to invasive illness.

During the bacteremic phase, the pneumococcus reaches and aims to survive in the bloodstream. In the blood, the pneumococcus confronts additional host defense mechanisms, the first line of defense being complement-mediated opsonization and phagocytosis. To survive, the pneumococcus increases its capsule size, reduces complement deposition on its surface and limits subsequent interaction with phagocytes (28,29). This is achieved by bacterial surface proteins that target specific complement components, thus inhibiting complement-mediated clearance, e.g., by recruiting factor H that inhibits the complement cascade (30,31). The second line of defense is recognition of the bacteria by antigen-presenting cells through the binding of pattern recognition receptors directed specifically toward general motifs of molecules expressed by the pathogen. This results in release of cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1 and IL-6 that further induce the recruitment of neutrophils and lymphocytes, and simultaneously cause cerebrovascular damage (32). A prominent risk resulting in vascular injury is exposure of blood to an extravascular tissue factor and activation of the coagulation cascade that may lead to vascular clot formation (33,34), BBB dysfunction and blood flow impairment (35).

Defense begins with the recognition of pathogen-associated molecular patterns (PAMPs), which are structural signatures characteristic of the bacteria. These include bacterial products such as proteins, lipids, nucleic acids and carbohydrates. PAMPs are initially sensed by antigen-presenting cells which express pattern recognition receptors. The main receptors involved in S. pneumoniae recognition are Toll-like 2 receptor (TLR-2, also known as CD282), which are recognized by peptidoglycans and lipoteichoic acids (68), TLR-4 (also known as CD284) that are recognized by exotoxin pneumolysin (69) and TLR-9 (also known as CD289), intracellular pattern recognition receptors that are activated by cytosine-guanosine motifs (CpG) in bacterial DNA (70). TLR-2 and −4 bind to myeloid differentiation factor 88 (MyD88) as a common intracellular adapter protein known, which activates the nuclear factor (NF)-κB pathway with subsequent upregulation of pro-inflammatory mediators. Studies have demonstrated that TLR-2 and −4 have a more prominent role than TLR 9 in the induction of the inflammatory response to pathogens and suggested that one receptor may compensate for the absence of the other (71). In addition, family members of the intracellular nucleotide oligomerization domain (NOD) -like receptors (NLRs), Nod-2, also have essential roles in Gram-positive peptidoglycan detection: When activated, they stimulate NF-κB or mitogen-activated protein kinase pathways and activate caspase-1 (72). This in turn stimulates the production of inflammatory cytokines and chemokines by astrocytes and microglia that express TLRs and NLRs (73). In humans, certain deficiencies and polymorphisms such as phosphorylation of interleukin receptor-associated kinase and Myd88 adapter protein have been associated with invasive pneumococcal disease such as meningitis (74).

As described by Del Rio Hortega, microglia comprise up to 20% of the non-neuronal cell population, are derived from the mesoderm and are considered as the resident macrophages of the CNS (75). The brain of adults has two major subsets of microglia: Parenchymal and perivascular microglia. The latter are located in the basal lamina of brain capillaries and the choroid plexus. The phagocytic properties of microglia (76) have been considered to be the first line of defense in the CNS and to impact numerous immune responses of the brain against infectious and acute as well as chronic neurological diseases (66,77). Resting microglia have small bodies and long, thin processes with ramified morphology, which correspond to the vigilant form which is able to promptly recognize homeostatic disturbances in the CNS (77). Under pathological conditions, microglia become activated and are characterized by an amoeboid morphology with short processes. Surface antigens and cytokine release corresponding to distinct phenotypes are associated with the transition from one form to another (78). The activated, phagocytic phenotype has been indicated to mediate the elimination of neurotoxic substances from brain parenchyma, such as blood-borne albumin (79). Since microglial cells are located in the perivascular space, it is likely that their interactions with endothelial cells influence the properties of the BBB. It has been suggested that, similarly to astrocytes, activation of microglia restores BBB integrity after its chemically stimulated loss by directing tight junction proteins to para-cellular domains (80). However, in neuro-inflammation, activated microglia may cause barrier impairment and BBB dysfunction by releasing the pro-inflammatory cytokine TNF-α (81). Apart from their participation in inflammatory and infectious events and their scavenger function, microglia also take part in several important physiological events in the adult brain, including induction of apoptosis in specific subpopulations of developing neurons, control of synaptogenesis and synaptic transmission as well as synthesis of neurotrophic factors (82).

Glial astrocytic cells are ideally situated to function as mediators in neurovascular communication; they surround synapses and can thus sense neuronal activity, whereas their end-foot processes envelop blood vessels and may signal (and sense) smooth muscle cells and/or pericytes (83). Astrocytes are enriched in potassium channels, purinergic receptors and the gap junction protein connexin 43, as well as the water-channel protein aquaporin-4 (84), indicating key roles in potassium buffering (85), calcium-dependent glio-vascular signaling and regulation of brain water content. Astrocytes release several vasoactive factors, including nitric oxide (NO) and arachidonic acid metabolites, having a prominent role as mediators of vasomotor activity (86). Moreover, astrocytes produce neutrophins and a wide range of anti-inflammatory cytokines (87). Upon activation of various signaling pathways [e.g. TLR/NLR and transforming growth factor (TGF) beta], astrocytes participate in innate immune reactions, synthesize and release neuro-inflammatory mediators, including several complement components, cytokines such as IL-1β and IL-6, and chemokines (88–92).

Following recognition of the pathogen and activation of non-neuronal cells, microglia and astrocytes produce and release a wide range of inflammatory molecules in the brain. These include molecules such as cytokines, free radicals, matrix metalloproteinases (MMPs) and chemokines, which contribute to an increase in BBB permeability and enable recruitment of leukocytes to the site of infection. Alongside bacterial elimination, this results with brain edema, increased intracranial pressure and impaired cerebral blood flow, and may lead to irreversible neuronal injury (88).

Cytokines are multifunctional pleiotropic proteins with crucial roles in cellular activation and cell-to-cell communication. Depending on their function, they are classified as being either pro- or anti-inflammatory according to the final balance of their effects on the immune system. Cytokines not only participate in the immune response but also in a variety of physiological and pathological processes, including events in the periphery and CNS; therefore, they act as immune regulators as well as neuromodulators. The neuro-immune interactions are bidirectional-cytokines can modulate the action, differentiation and survival of neurons, while neurotransmitters and neuropeptides released from neurons have a central role in influencing the immune response. The various cytokines directly affecting the CNS either originate from the peripheral immune system, in which case they migrate across the opened BBB, or they are produced locally within the CNS by brain resident cells (89).

Following pneumococcal infection, release of pro-inflammatory cytokines includes molecules such as IL-1, IL-6, TNF-α and interferon (IFN)-γ. These cytokines are released by endothelial cells, astrocytes, microglia and neurons, resulting in increased BBB permeability and recruitment of leukocytes from the circulatory blood (32,90). TNF-α is a 158 amino acid cytokine; it was found to be produced in the cortex and hippocampus during the first 6 h, and remained elevated until 96 h after meningitis initiation (91,92). TNF-α binding to its cognate receptor leads to NF-κB activation that regulates the expression of other pro-inflammatory mediators (93). In patients with bacterial meningitis, intrathecal levels of TNF-α were correlated with the severity of BBB disruption, neurologic sequelae and disease severity (58). However, TNF-α deficient mice infected with S. pneumoniae demonstrated increased mortality and spatial memory deficits (94), suggesting that TNF-α reflects disease severity but is not required to induce neurological complications. IL-1β is a pro-inflammatory cytokine produced early after bacterial invasion of the brain in the cortex and hippocampus by perivascular mononuclear phagocytes, macrophages and glial cells through stimulation of bacterial compounds or TNF-α (91,92). IL-1β increases the expression of nearly all other cytokines, including TNF-α, IL-6, IFN-γ and chemokines; however, its role in bacterial meningitis remains elusive. In patients with bacterial meningitis, IL-1β levels were not correlated with the degree of BBB opening (58). By contrast, decreased levels of IL-1β were associated with lower intracranial pressure (ICP), leukocyte recruitment and brain edema (95). Intrathecal administration of IL-1β did not lead to CSF pleocytosis or brain edema, but administration of anti-IL-1β antibodies decreased TNF-α-induced leukocyte influx (96). IL-1 receptor (IL-1R) gene-deficient mice succumbed earlier to the disease and their mortality rate was significantly elevated, indicating that endogenous IL-1β is required for an adequate host defense in pneumococcal meningitis (97). IL-6 is expressed mostly in the cortex and is produced by endothelial cells, astrocytes and monocytes (98). Although the effects of IL-6 are predominantly pro-inflammatory, including leukocyte recruitment, the potent induction of acute-phase proteins and fever (99), it also acts as an anti-inflammatory cytokine. Indeed, IL-6 gene deficiency in mice with bacterial meningitis was found to be associated with an increased inflammatory response and an impaired defense against pneumococcal pneumonia, as well as reduced vascular permeability and ICP (100,101). IFN is another important pro-inflammatory mediator in pneumococcal meningitis, found at elevated concentrations in meningitis patients' CSF (102). It has been suggested that IFN-γ is produced following bacterial recognition through activation of the NLR inflammasome pathways (103,104).

Anti-inflammatory cytokines such as IL-10 and TGF-β have been shown to be upregulated during pneumococcal meningitis. IL-10 is a potent immune-suppressive cytokine produced by brain cells such as neurons and microglia as well as macrophages and monocytes (105), and elevated levels have been found in the CSF of patients with bacterial meningitis (106). IL-10 was shown to inhibit the production of pro-inflammatory cytokines including TNF-α and IL-6, as well as the release of reactive oxygen species (ROS) (107), and to induce impairment of neutrophil phagocytosis and killing (108). Accordingly, systemic administration of recombinant IL-10 in a rat model of pneumococcal meningitis resulted in lower levels of pro-inflammatory cytokines, CSF pleocytosis and cerebral edema (107). However, in IL-10 knockout mice with pneumococcal meningitis, bacterial loads and survival rates were similar to those in wild-type mice (97). TGF-β is a pleiotropic cytokine with potent inflammatory regulatory activity expressed in neurons and glial cells. Among its multiple functions, it modulates T-cell activity, including proliferation and differentiation processes (109,110). The influence of TGF-β on the immune reactivity of the CNS following infection remains to be under debate; it suppresses the production of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α from microglia and macrophages (111,112), but has also been reported to increase the production in cultured astrocytes (113). Absence of TGF-β signaling was demonstrated to facilitate the recruitment of leukocytes and the clearance of S. pneumoniae in the CNS of mice with meningitis, resulting in reduced cerebrovascular complications (114).

Chemokines are a subgroup of cytokines, which are considered to have chemotactic activity due to their ability to induce directed chemotaxis in nearby responsive cells. In pneumococcal meningitis, multiple chemokines have been reported to be upregulated and enhance the recruitment and accumulation of inflammatory cells in the CSF (115). IL-8 is produced by a wide range of cells, including macrophages and monocytes, through IL-1β, TNF-α and stimuli of live bacteria. In rabbits with pneumococcal meningitis, intravenous administration of a monoclonal antibody to IL-8 attenuated pleocytosis (116). Another group of chemokines are the CC chemokine ligands (117), including monocyte chemoattractant proteins and macrophage inflammatory proteins (MIPs), which are released by astrocytes and microglia (118,119). Intracisternal administration of recombinant molecules was found to induce BBB dysfunction, CSF leukocytosis and brain edema (96).

As part of the immune response to bacterial infection, blood-derived leukocytes, such as neutrophilic granulocytes and monocytes, enter the CNS to clear the pathogen. The recruitment of leukocytes through the BBB is associated with meningeal and perivascular macrophage activation and upregulation of the endothelial adhesion molecules selectin and integrin (120,121). The PAF is a protein which facilitates the adhesion of leukocytes to the endothelium and is produced by neutrophils and endothelial cells in response to inflammatory stimulation (122). Another translocation mediator in pneumococcal meningitis is the urokinase-type plasminogen activator, which is also considered as a fibrin degrader (123). However, after migration into the CNS, leukocytes release a variety of toxic molecules (e.g., ROS), causing cerebrovascular complications and neuronal injury (115).

MMPs are a family of neutral proteases which are of importance in normal development, wound healing and a large variety of pathological processes, including neuro-inflammation and the spread of metastatic cancer cells (124). In the CNS, MMPs have been shown to degrade components of the basal lamina, leading to BBB breakdown, and contribute to inflammation in numerous neurological diseases. In response to cellular stress, MMPs are secreted by a wide range of cells, including activated neutrophils and macrophages, neurons and glial cells (125). High concentrations of MMP-8 and −9 have been detected in the CSF of patients with bacterial meningitis, and indeed, high concentrations of MMP-9 have been demonstrated to be correlated with TNF-α levels, to induce BBB dysfunction, and to be a risk factor for the development of post-meningitis neurological deficits (125).

A plethora of studies on patients as well as animal models implied that free radicals, including ROS and reactive nitrogen species (RNS), hydrogen peroxide (H₂O₂) and hydroxyl peroxide have a central role in the development of intracranial complications and brain damage in bacterial meningitis (126,127). Brain cells and attracted leukocytes produce free radicals as part of the host immune response to invasive bacterial infection (128,129). S. pneumoniae itself is also an important source of H₂O₂, which causes direct cytotoxic damage and also reacts with the host's NO to form peroxynitrite (ONOO⁻), a highly reactive oxidant (130). ONOO⁻ is formed at sites where NO and superoxide anions are produced simultaneously (131,132). ONOO⁻ can be cytotoxic by a number of mechanisms, including tyrosine nitration that affects cellular signaling (133), lipid peroxidation that induces loss of membrane function and integrity (134), and production of cytokines and MMPs (135,136). Adjuvant therapy with an ONOO⁻ scavenger reduces the number of CSF leukocytes as well as IL-1β and MIP-2 concentrations in the brain (137), which suggests that ROS/RNS and pro-inflammatory chemokines/cytokines are involved in the attraction of blood-bound leukocytes into the subarachnoid space (138). Furthermore, treatment with antioxidants was shown to attenuate BBB leakage (137,139).

An important histopathological finding in patients with S. pneumoniae meningitis as well as in experimental meningitis is cell death in the dentate gyrus of the hippocampus (140), which was also correlated with the development of learning deficits (141,142). S. pneumoniae is able to induce two functionally distinct forms of programmed cell death in the brain, which proceed either via TLR-dependent or -independent pathways. The pneumococcal cell wall is a pro-inflammatory component and causes apoptosis in the hippocampus mediated via TLR-2 and activation of caspases (143). Living pneumococci and the major cytotoxins pneumolysin and H₂O₂ appear to induce damage to endothelial cells of the BBB through a TLR-independent pathway (143). The these toxins induce an increase in ROS and intracellular calcium, resulting in mitochondrial dysfunction that leads to the release of apoptosis-inducing factors into the cytosol (130,144).

Pneumococcal meningitis is at times accompanied by cerebrovascular complications, including ischemic stroke, venous thrombosis, intracerebral hemorrhage and vasculitis (145–148). Additional studies have suggested that vasospasm, thrombosis and diffused cerebral intravascular coagulation may each contribute to cerebral injury (149–151). Other factors, including alterations in blood pressure and impaired cerebral auto-regulation of blood flow may also have a role (152,153). Following bacterial infection and stroke, cells in the penumbra are subjected to various inflammatory components such as infiltrating leukocytes, pro-inflammatory cytokines and free radicals, which may be deleterious and lead to further cellular damage (154). Furthermore, subcortical ischemic white matter lesions have been identified, which led to axonal injury (155). Recently, the pro-inflammatory cytokine IL-1 was identified as a key mediator of pneumococcal infection-induced cerebral ischemia and neuronal injury (156). In a model of ischemic stroke, bacterial aspiration led to severe pneumonia, which persisted for fourteen days after stroke induction, suggesting that stroke increases the susceptibility to infection (157). Another severe vascular complication in pneumococcal meningitis is venous thrombosis (158,159), associated with impairments of blood flow and perfusion pressure, intracranial hemorrhage, BBB dysfunction and cerebral edema (160,161).

Seizures are a common complication of S. pneumoniae meningitis during the acute phase of illness, with an increased risk of developing unprovoked seizures (e.g., epilepsy) later in life (162,163). Seizures occur frequently in adult patients and are associated with severe inflammation and structural CNS lesions (164,165). In children, the probability of the occurrence of status epilepticus associated with fever is higher than that of short febrile seizures, and the classical symptoms and signs of meningitis may be absent under such conditions (166,167).

Studies have suggested that cerebrovascular injury and BBB dysfunction, which allow the infiltration of albumin, lead to activation of TGF-β signaling in astrocytes. This further results in a neuro-inflammatory response associated with up-regulation and release of IL-1β, IL-6 and TGF-β cytokines and seizures (168–172). Thus, in patients with pneumococcal meningitis, increased BBB permeability and inflammatory response as described above may similarly increase the likelihood of seizures.

One of the most common sequelae of pneumococcal meningitis is bacterial labyrinthitis and sensory neural hearing loss. Labyrinthitis occurs as the bacteria reach the cochlear aqueduct from the subarachnoid space or travel with the eighth cranial nerve in the internal auditory canal (173,174), contributing to hair cell injury and neuronal cell death (175). A study using a mouse model of pneumococcal meningitis suggested a pro-inflammatory role for the TLR-MyD88 signaling pathway as a trigger for labyrinthitis (176). Synthesis of TNF-α and free radicals has been demonstrated to have an important role in hearing loss following infection, and inhibition of their production was found to have a protective effect (175,177).

Mild to severe intellectual and behavioral deficits, including cognitive impairment, learning disabilities and attention deficit hyperactivity disorder are well-recognized complications of bacterial meningitis in children and are the most common long-term sequelae (178,179). Psychiatric illness and neurodegenerative diseases have been linked with cerebrovascular damage and BBB alterations: An elevated CSF/serum albumin ratio in patients suffering from dementia compared to non-demented individuals was found (180), and elevated serum levels of S100 calcium-binding protein B, normally found exclusively in the brain, were demonstrated in patients suffering from depression and schizophrenia (181). In addition, accumulating evidence indicated that immunologic responses have a role in deficits in cognitive function as well as depression; Increases in pro-inflammatory cytokines, including TNF-α, IFN and IL-1β and −6, alongside a relative reduction of the anti-inflammatory cytokine IL-10, were demonstrated in depression. Furthermore, a positive correlation was shown between plasma concentrations of inflammatory mediators, such as IL-1β and IL-6, and the severity of depression symptoms (182,183). These suggest that early vascular injury during meningitis, including BBB dysfunction and neuroinflammatory processes, can be associated with delayed neuropsychological and mental problems.